Oligopeptide microarrays are widely used in research and healthcare. Within these areas, oligopeptide microarrays are suitable for many different applications. Oligopeptide microarrays for example provide a tool for the identification of biologically active motifs, e.g., oligopeptide microarrays may imitate potential active motifs of ligands for screening the binding to corresponding receptors. Furthermore, the oligopeptide microarrays might reflect specific sequences of disease associated antigens. Such oligopeptide microarrays can be utilized to detect antibodies from patient samples suggesting the presence of certain inflammatory diseases and infections. Another important application of the oligopeptide microarrays is the discovery of biochemical interactions, including the binding of proteins or DNA. Oligopeptide microarrays can further be used for profiling cellular activity, enzymatic activity, cell adhesion, and the like.
Different methods for the production of oligopeptide microarrays are known in the state of the art. Spotting prefabricated peptides or in-situ synthesis by spotting reagents, e.g., on membranes, prevail. One of the most commonly used methods to generate peptide arrays of higher density are the so-called photolithographic techniques, where the synthetic design of the desired biopolymers is controlled by suitable photolabile protecting groups (PLPG) releasing the linkage site for the respective next component (amino acid, oligonucleotide) upon exposure to electromagnetic radiation, preferably light (Fodor et al., (1993) Nature 364:555-556; Fodor et al., (1991) Science 251:767-773).
Two different photolithographic techniques are known in the state of the art: 1) A photolithographic mask is used to direct light to specific areas of the synthesis surface effecting localized deprotection of the PLPG. The drawback of this technique is that a large number of masking steps are required resulting in a relatively low overall yield and high costs, e.g., the synthesis of a peptide of only six amino acids in length could require over 100 masks. 2) The second technique is the so-called maskless photolithography, where light is directed to specific areas of the synthesis surface effecting localized deprotection of the PLPG by digital projection technologies, such as micromirror devices (Singh-Gasson et al., Nature Biotechn. 17 (1999) 974-978). Thus, time-consuming and expensive production of exposure masks is eliminated.
The use of PLPG, providing the basis for the photolithography based synthesis of oligopeptide microarrays, is well known in the art. Commonly used PLPG for photolithography based biopolymer synthesis are for example α-methyl-6-nitropiperonyl-oxycarbonyl (MeNPOC) (Pease et al., Proc. Natl. Acad. Sci. USA (1994) 91:5022-5026), 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC) (Hasan et al. (1997) Tetrahedron 53: 4247-4264), nitroveratryloxycarbonyl (NVOC) (Fodor et al. (1991) Science 251:767-773) and 2-nitrobenzyloxycarbonyl (NBOC) (Patchornik et al. (1970) 21:6333-6335).
Amino acids have been introduced in photolithographic solid-phase peptide synthesis of oligopeptide microarrays, which were protected with NPPOC as a photolabile amino protecting group, wherein glass slides were used as a support (U.S. Patent Publication No. 2005/0101763 A1). The method using NPPOC protected amino acids has the disadvantage that the half-life upon irradiation with light of all (except one) protected amino acids is within the range of approximately 2 to 3 minutes under certain conditions. In contrast, under the same conditions, NPPOC-protected tyrosine exhibits a half-life of almost 10 minutes. As the velocity of the whole synthesis process depends on the slowest sub-process, this phenomenon increases the time of the synthesis process by a factor of 3 to 4. Concomitantly, the degree of damage by photogenerated radical ions to the growing oligomers increases with increasing and excessive light dose requirement.
The object of the present invention is, therefore, the provision of a high density oligopeptide microarray with high sensitivity and a method for its synthesis not showing the above-described drawbacks. Thus, the object of the present invention is the provision of a sensitive oligopeptide microarray of high quality, which can be synthesized within a significantly shorter time period as compared to the state of the art, consequently reducing costs of production of oligopeptide microarrays.